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Abstract

Background

Glutamate excitotoxicity is thought to be involved in the pathogenesis of neurodegenerative
disease. One potential source of glutamate is N-acetyl-aspartyl-glutamate (NAAG) which
is hydrolyzed to glutamate and N-acetyl-aspartate (NAA) in a reaction catalyzed by
glutamate carboxypeptidase (GCP). As a result, GCP inhibition is thought to be beneficial
for the treatment of neurodegenerative diseases where excess glutamate is presumed
pathogenic. Both pharmacological and genetic inhibition of GCP has shown therapeutic
utility in preclinical models and this has led to GCP inhibitors being pursued for
the treatment of nervous system disorders in human clinical trials. Specifically,
GCP inhibitors are currently being developed for peripheral neuropathy and neuropathic
pain. The purpose of this study was to develop a pharmacodynamic (PD) marker assay
to use in clinical development. The PD marker will determine the effect of GCP inhibitors
on GCP enzymatic activity in human skin as measure of inhibition in peripheral nerve
and help predict drug doses required to elicit pharmacologic responses.

Methods

GCP activity was first characterized in both human skin and rat paw pads. GCP activity
was then monitored in both rodent paw pads and sciatic nerve from the same animals
following peripheral administration of various doses of GCP inhibitor. Significant
differences among measurements were determined using two-tailed distribution, equal
variance student's t test.

Results

We describe for the first time, a direct and quantifiable assay to evaluate GCP enzymatic
activity in human skin biopsy samples. In addition, we show that GCP activity in skin
is responsive to pharmacological manipulation; GCP activity in rodent paws was inhibited
in a dose response manner following peripheral administration of a potent and selective
GCP inhibitor. Inhibition of GCP activity in rat paw pads was shown to correlate to
inhibition of GCP activity in peripheral nerve.

Conclusion

Monitoring GCP activity in human skin after administration of GCP inhibitors could
be readily used as PD marker in the clinical development of GCP inhibitors. Enzymatic
activity provides a simple and direct measurement of GCP activity from tissue samples
easily assessable in human subjects.

Background

Excess glutamate has been shown to be neurotoxic in many degenerative diseases of
the central and peripheral nervous system [1]. One potential source of glutamate is N-acetyl-aspartyl-glutamate (NAAG), a dipeptide
found in the brain and peripheral nerves [2]. Glutamate carboxypeptidase (GCP) catalyzes the hydrolysis of NAAG to glutamate and
N-acetyl-aspartate (NAA) [3]. There are two known GCP enzymes in the nervous system with similar pharmacological
profiles: GCPII and GCPIII. GCPII, the more widely studied homolog, exhibits a high
level of expression and it is found on the cell surface of astrocytes and non-myelinating
Schwann cells [4-6]. GCPIII message on the other hand, is expressed in mouse cortical and cerebellar
neurons in culture [7]. Inhibition of the GCP-catalyzed reaction should be beneficial for the treatment
of degenerative diseases associated with excess glutamate. In fact, both genetic and
pharmacological inhibition of GCP has been found to be neuroprotective in a variety
of cell and animal models of disease involving excess glutamate [8-17]. Based on these data, GCP inhibitors are currently being pursued in the clinic as
therapeutics for the treatment of peripheral neuropathy and neuropathic pain [18].

Clinical development of a drug can be aided by pharmacodynamic (PD) marker assays
to predict drug doses required to elicit pharmacologic responses. Until recently,
monitoring NAAG levels in biological matrices (e.g. CSF, plasma, and urine) was considered
the PD marker of choice to monitor GCP inhibition [19]. For clinical studies, the best biological matrix to evaluate CNS/PNS penetration
is cerebrospinal fluid. However, sample collection requires considerable skill and
it is uncomfortable to patients. In addition, NAAG measurements involve the use of
HPLC or LC-MS/MS [19] and are only a surrogate marker of enzyme inhibition. Quantifying GCP enzymatic activity
on the other hand, provides a direct measurement for monitoring enzyme inhibition
and is relatively straightforward to carry out. Until recently, GCP activity measurements
were thought to be unfeasible as PD marker assays in the clinic because GCP was thought
to be present only in nervous tissue, prostate, intestinal tract, and kidney, tissues
that are not easily accessible for collection during clinical studies [20]. However, local administration of GCP inhibitors have been shown to be analgesic
in peripheral pain in rats [21] and NAAG is known to be synthesized and localized in spinal sensory ganglia [22]. Further, GCP is located in Schwann cells [4,5] which exist in the epidermis [23]. Consequently, we set out to determine if GCP was measureable in human skin. In this
report, we describe for the first time, quantifiable GCP activity in human skin biopsy
samples. Further, to determine if GCP activity in skin is amenable to pharmacological
manipulation, we conducted rodent studies on GCP activity in rat paws after dosing
with GCP inhibitor. We report robust GCP activity in rodent paws which is sensitive
to inhibition in a dose response manner following peripheral administration of a GCP
inhibitor. Further, inhibition of GCP activity in rodent paws was shown to correlate
to GCP inhibition in peripheral nerve.

Methods

Human skin biopsy collection

Punch skin biopsies (3 mm) were obtained from the distal thigh of healthy volunteers
after anesthesia with 0.5 cc 2% lidocaine subcutaneous injection [24]. The protocol was approved by the Johns Hopkins Institutional Review Board in compliance
with the Helsinki declaration. Samples were placed in cold Tris buffer (pH 7.4) and
GCP enzymatic activity was carried out within 1 h of collection.

Rodent drug dosing and paw and sciatic nerve sample collection

All experimental protocols were approved by the Institutional Animal Care and Use
Committee of SoBran, Inc., Baltimore and adhered to all of the applicable institutional
and governmental guidelines for the humane treatment of laboratory animals. Rats (male
Wistar) were administered vehicle (HEPES saline, pH 7, 50 mM) or 2-PMPA (1, 10 and
100 mg/kg, i.p.) using a dosing volume of 2 mL/kg. There were 10 animals in each group.
Animals were sacrificed 1 h after 2-PMPA or vehicle administration. 2-PMPA brain concentrations
were previously shown to be highest 50 - 75 min after i.p. administration [12]. Skin was collected from the planter hindpaw by 3 mm skin biopsy dissection and stored
at -80°C until ready for analysis. In order to obtain sciatic nerve, 1-2 cm incisions
were made on the skin on top of the mid thigh so that sciatic nerve, gluteus superficialis
muscle and biceps femoris muscle became exposed. The three were then separated and
5 mm of sciatic nerve was dissected out.

Human skin biopsy and rodent paw and sciatic nerve sample preparation

Human skin biopsies were sonicated in Tris buffer (pH 7.4, 40 mM, 0.5 mL) for 1 min
in ice. The mixture was centrifuged for 2 min at 16000 × g; the supernantant (containing
cytosolic fraction) was removed and the resulting pellet (containing plasma membrane)
was reconstituted in 70 μL assay buffer (Tris pH 7.4, 40 mM containing 1 mM CoCl2) and used as source of GCP in the activity assay. Rat paw pads and sciatic nerve
isolated from vehicle and 2-PMPA treated animals were sonicated for 2 min in ice.
The mixture was centrifuged for 2 min at 16000 × g and the resulting pellet was reconstituted
similar to the pellets obtained from the human skin dissections.

GCP activity measurements were carried out following published procedures [3,25]. Briefly, the reaction mixture contained [3H]-NAAG (70 nM, 50 Ci/mmol) and reconstituted pellet (human skin, paw pad, or sciatic
nerve) in Tris-HCl containing 1 mM CoCl2 in a total volume of 90 μL. The reaction was carried out at 37°C at different times
as indicated, and stopped with ice-cold sodium phosphate buffer (pH 7.4, 0.1 M, 90
μL). When human skin was used as GCP source, the reaction was carried out in the presence
and absence of the selective GCP inhibitor 2-PMPA (1 μM). When rat tissue was used
from the ex vivo study, 2-PMPA was administered i.p. and the animals were sacrificed and their paw
pads removed for GCP enzymatic determinations. In both cases, blanks were obtained
by incubating the reaction mixture without pellet. Duplicate aliquots of 90 μL from
each terminated reaction was transferred to a well in a 96-well spin column containing
AG1X8 ion- exchange resin; the plate was centrifuged at 1000 rpm for 5 minutes using
a Beckman GS-6R centrifuge equipped with a PTS-2000 rotor. [3H]-NAAG bound to the resin and [3H]-glutamate eluted in the flow through. Columns were then washed twice with formate
(1 M, 90 μL) to ensure complete elution of [3H]-glutamate. The flow through and the washes were collected in a deep 96-well block;
from each well with a total volume of 270 μL, a 200 μL aliquot was transferred to
a glass scintillation vial, to which 10 ml of Ultima-Gold (Perkin Elmer) was added.
The radioactivity in each vial corresponding to [3H]-glutamate was determined via a Beckman LS-6000IC scintillation counter. Radioactivity
values in dpm were converted to fmoles of glutamate using the relation 1 pCi/2.2 dpm
and the specific activity of [3H]-glutamate (same as that of [3H]-NAAG: 1 fmole/50 pCi). As a result, if 16711 dpm [3H]-glutamate were measured after incubating 10 mg tissue for 1 h, the normalized activity
would be: 16711 dpm × (1 pCi/2.2 dpm) × (1 fmole/50 pCi)/10 mg tissue = 15 fmole/h/mg
tissue.

Statistical Analysis

Determination of 2-PMPA concentration in rodent paws by LC-MS/MS

Frozen samples were thawed in a water bath at ambient temperature and subjected to
a liquid extraction using MeOH. Samples were placed in brown glass vials containing
500 μL of 100% MeOH. The vial was capped and mixed vigorously for 10 sec on a vortex-mixer
followed by 30 min on an automated multitude shaker, followed by incubation for 24
h at 4°C. The top organic layer was transferred to a disposable borosilicate glass
culture tube (13 × 100 mm) and evaporated to dryness at 40°C under a gentle stream
of nitrogen. The residue was reconstituted in 100 μL acetonitrile-water (1:1, v/v)
containing the internal standard, temazepam (50 μg/mL), by vortex mixing (30 sec)
and immersion in an ultrasound bath (5 min). The sample was transferred to a 250 μL
polypropylene auto sampler vial sealed with a Teflon crimp cap, and a volume of 50
μL was injected onto the HPLC instrument for quantitative analysis using a temperature-controlled
auto sampling device operating at 10°C.

Chromatographic analysis was performed using a Waters ACQUITY UPLC (Milford, MA, USA).
Separation of the analytes from potentially interfering material was achieved at ambient
temperature using a Waters Altantis column (100 × 2.1 mm i.d.) packed with a 3 μm
ODS stationary phase, protected by a guard column packed with 3.5 μm RP18 material
(Milford, MA, USA). The mobile phase used for the chromatographic separation was composed
of acetonitrile-water (60:40, v/v) containing 0.1% formic acid, and was delivered
isocratically at a flow rate of 0.3 mL/min. The column effluent was monitored using
an AB SCIEX TRIPLE QUAD 5500 triple-quadrupole mass-spectrometric detector (Applied
Biosystems, Foster City, CA, USA). The instrument was equipped with an electrospray
interface, operated in a positive mode and controlled by the Analyst version 1.5 software
(Applied Biosystems). The spectrometer was programmed to allow the [MH+] ion of 2-PMPA at m/z 226.8 and that of the internal standard at m/z 301.1 pass through
the first quadrupole (Q1) and into the collision cell (Q2). The daughter ions for
2-PMPA (m/z 191.1) and the internal standard (m/z 255.1) were monitored through the
third quadrupole (Q3). Calibration curves were generated over the range of 200 to
10,000 ng/mL. Mouse paw pad samples were then quantitated in μg/g as: nominal concentration
(ng/mL) × 0.0625 (standardized dilution) × sample weight (in mg).

Results and Discussion

GCP II activity is present in human skin biopsies

Skin biopsies from human volunteers were homogenized, the homogenate was centrifuged
and the pellet was used as source of GCP in the enzyme activity assay. Reconstituted
pellet was then incubated with [3H] NAAG and production of glutamate was determined in the presence and absence of
2-PMPA, a highly selective GCP inhibitor (Methods) [26]. When pellets obtained from human skin biopsy were used, conversion to glutamate
was 11 ± 0.2 fmole glutamate generated/h/mg tissue. GCP activity monitoring in human
skin was attempted previously, but reported to exist below the limit of detection
[27]. In this study, according to previous findings [27], we found that homogenate preparations of human skin exhibited a very low GCP activity
that was difficult to measure. However, when using pellet preparations (methods) as
source of GCP, we found significant measurable activity in human skin biopsies that
was inhibited by 90% when 2-PMPA, a highly specific GCP inhibitor, was added to the
assay mixture.

A time course of glutamate production after different incubation times (0.5, 1, 2,
3, 5, 7.5, 14, 18 and 24 h) was carried out. Due to the limited number of samples
that can be obtained from one person at a time, samples from different patients were
used in this study. Consequently, each time point was an independent determination;
pellets were prepared from separate skin biopsies from different volunteer donors
over two separate days. GCP activity was found to be linear for the first 7.5 h of
incubation (Figure 1). [3H]-NAAG at 70 nM (~770,000 dpm) provided robust sensitivity to follow GCP activity;
there were approximately 5,000 and 80,000 dpm of [3H]-glutamate after 0.5 and 7.5 h incubation respectively. These values corresponded
to 0.6 and 10% conversion of reactant to product indicating that sufficient substrate
concentration was used and that if additional GCP activity had been present, additional
activity would have been observed. The linear relationship with respect to time using
samples from different donors suggests that GCP levels among normal volunteers are
relatively similar.

Figure 1.Dependence of GCP activity in human skin biopsy on time of incubation - Human skin biopsies were sonicated for 2 min in ice. The resulting mixture was centrifuged
at 16000 × g; precipitate from each preparation was used as GCP source in the activity
assay. Incubations with [3H] NAAG (70 nM) at 37°C were carried out at 0.5, 1, 2, 3, 5, 7.5, 14, 18 and 24 h.
Time points correspond to incubations carried out with biopsies obtained from different
donors. Major plot illustrates the correspondence of enzyme activity ([3H]-glutamate production in dpm) with time while linearity was observed. Inset illustrates
GCP activity measured at times up to 24 h.

GCP activity is present in rodent paw pads

A parallel determination of GCP activity was carried out using male Wistar rat paw
pads. Reconstituted pellet preparations from rat paw pads were used as source of GCP
II and incubated with [3H] NAAG. The amount of GCP activity in rat paw pads was found to be 15 ± 0.2 fmole
glutamate generated/h/mg tissue). Interestingly, the amount of GCP activity found
in rat paw pads (15 ± 0.2 fmole/h/mg tissue) was similar to that obtained from human
skin (11 ± 0.2 fmole/h/mg tissue).

To be useful as clinical PD marker, GCP activity in skin needs to be amenable to inhibition
by peripheral administration of GCP inhibitors in a dose response manner. In order
to determine if GCP activity in paw pads in vivo could be inhibited by peripheral administration of 2-PMPA, rats were treated with
1, 10 and 100 mg/kg 2-PMPA (i.p.) as well as vehicle control. Animals were sacrificed
1 h after compound administration, paw pads isolated and GCP activity determined (Methods).
GCP activity in paw pad preparations from animals treated with 1 mg/kg 2-PMPA was
similar to that of controls. On the other hand, paw pads from animals treated with
10 and 100 mg/kg exhibited significantly reduced GCP activity: 60 ± 11 and 47 ± 11%
respectively when compared to control animals (Figure 2A). Importantly, these are the doses of 2-PMPA previously shown to exhibit therapeutic
benefit [13].

Peripheral administration of 2-PMPA inhibits GCP activity in sciatic nerve in a dose
response manner and it correlates to inhibition observed in rat paw pads

Sciatic nerve is the target tissue for GCP inhibitors in clinical trials for peripheral
neuropathy and neuropathic pain. Consequently, it is important to demonstrate that
there is a correlation of GCP inhibition in skin and peripheral nerve after administration
of different doses of GCP inhibitor. GCP activity in sciatic nerve preparations from
animals treated with 1, 10 and 100 mg/kg 2-PMPA was reduced to 92 ± 11, 35 ± 6 and
10 ± 4% respectively compared to activity in sciatic nerve isolated from control animals
(Figure 2B). Albeit to a different extent, GCP inhibition in sciatic nerve is attained at similar
2-PMPA doses (10 and 100 mg/kg i.p.) as in footpad tissue. Taken together, these results
suggested that it will be possible to follow GCP inhibition in the skin as a marker
of GCP inhibition in peripheral nerve.

2-PMPA is measurable in rat paw pads

Given that inhibition of GCP was observed in paw pads, we wanted to confirm the presence
of 2-PMPA in paw pads after peripheral administration of 2-PMPA. Animals were given
2-PMPA (100 mg/kg, i.p.), sacrificed 1 h after compound administration and paw pads
isolated for direct determination of 2-PMPA levels by LC-MS/MS (Methods). Since 2-PMPA
detection by mass spectrometry has low sensitivity due to ion suppression, we chose
to analyze samples from animals that had received 100 mg/kg 2-PMPA rather than 10
mg/kg to increase the probability of detecting 2-PMPA. The characteristic fragmentation
pattern for 2-PMPA was readily detected (Figure 3A) and the chromatographic peaks of 2-PMPA and internal standard (Figure 3B) allowed for quantitation of material in the sample. Paw pads from animals that were
treated with compound showed 38 ± 5 μg/g tissue (n = 9) (Figure 3B) a concentration high enough to inhibit GCP activity [25] while the compound was undetectable in paw pads isolated from vehicle-treated animals.

Conclusions

As a biomarker of GCP inhibition in the clinic, skin biopsy measurements of GCP activity
has three areas of improvement over the prior NAAG bioassay including simpler sample
collections, less expensive and time consuming sample analyses, and the ability to
quantitate direct vs. indirect measurement of GCP activity. Sample collection for
NAAG bioanalysis involves CSF collection which requires considerable skill and can
be uncomfortable to patients; the newly described procedure uses skin biopsies which
is readily accessible and can be collected multiple times from a single subject permitting
the ability to evaluate GCP activity before and after administration of the drug.
NAAG analysis uses mass spectrometry which requires a specialized laboratory and expensive
instrumentation. The new procedure monitors GCP activity in the skin ex vivo by following the conversion of [3H]-NAAG to [3H] glutamate in a simple enzymatic assay that can be carried out in a standard biochemistry
laboratory. Finally, the older procedure involved measurements of NAAG levels as surrogate
markers of GCP activity; the new procedure monitors GCP enzymatic activity directly.
In short, monitoring of GCP activity in human skin after administration of GCP inhibitors
can be readily utilized as a PD marker in the clinical development of GCP inhibitors.
The activity assay provides a simple and direct measurement of GCP activity from tissue
samples easily assessable in human subjects.

Competing interests

CR, MS and BSS are former Eisai employees; Eisai is currently working on the development
of a GCP inhibitor.

Authors' contributions

CR helped with study design and writing of the manuscript. MS carried out GCP activity
measurements in the different biological matrices. MP and GJE organized the collection
of human skin. MAR and MZ carried out 2-PMPA analysis by LC-MS/MS. BSS conceived the
study and study design and guided the writing and editing of the manuscript. All authors
read and approved the final manuscript.

Acknowledgements and Funding

This work was supported in part by the Analytical Pharmacology Core of the Sidney
Kimmel Comprehensive Cancer Center at Johns Hopkins (NIH grants UL1 RR025005; MAR
and MZ), the Shared Instrument Grant (1S10RR026824-01; MAR), and the Juvenile Diabetes
Research Foundation (MP, RO, and GE).

References

Doble A: The role of excitotoxicity in neurodegenerative disease: implications for therapy.